The peptide-tagged recombinant domains are also indicated under the sequence with the corresponding fluorophore colors used for detection in the reversed FluoroSpot assay. The relevant domain boundaries are indicated with amino acid positions under each schematic. ( C) Schematic structure of the VAR2CSA variants included in this study, depicting the N-terminal segment (NTS), the six Duffy binding-like (DBL) domains, the interdomain (ID) regions, the transmembrane domain (TM), and the intracellular acidic terminal segment (ATS). ( B) In ‘apparent’ cross-reactivity a VAR2CSA polymorphic epitope is recognized by several different variant-specific monoclonal antibodies present in serum. ( A) Schematic representing ‘true’ cross-reactivity where an epitope shared among different VAR2CSA variants (V1–V4) is recognized by a single monoclonal antibody (colored in black) present in the polyclonal serum. A major obstacle is the inability to separating true (i.e., a single antibody that recognizes a conserved epitope shared by multiple allelic variants Figure 1A) from apparent cross-reactivity (multiple antibodies, each recognizing a variant-specific epitope Figure 1B), because this requires analysis at the single B-cell level. However, it is a goal that is difficult to achieve using conventional approaches such as analysis of immune sera by enzyme-linked immunosorbent assay (ELISA).
Consequently, the identification of conserved and functionally important antibody epitopes in key antigens is a major goal of malaria immunology research. The extensive inter-clonal polymorphism and intra-clonal variation of key parasite antigens appear to be important reasons. However, acquisition of protection following natural exposure takes years to develop, and complete protection is rarely if ever achieved (reviewed by Hviid, 2005). falciparum malaria following natural exposure is mediated mainly by IgG antibodies with specificity for the asexual blood stages of the infection ( Cohen et al., 1961 Sabchareon et al., 1991). This parasite alone was responsible for an estimated 241 million disease episodes and 627,000 deaths in 2020, mainly in sub-Saharan Africa ( WHO, 2021).Īcquired immunity to P. At least five species of these parasites can cause disease in humans, but by far the most serious is Plasmodium falciparum.
Malaria is a serious infectious disease caused by mosquito-transmitted protozoan parasites of the genus Plasmodium. The assay is adaptable to the analysis of other polymorphic antigens, rendering it a powerful tool in studies of immunity to malaria and many other diseases. Using VAR2CSA-type PfEMP1-a notoriously polymorphic antigen involved in the pathogenesis of placental malaria-as a model, we demonstrate the robustness of the assay and its applicability to analysis of true cross-reactivity of monoclonal VAR2CSA-specific antibodies in naturally exposed individuals. In this study, we present a further enhancement of this assay that makes direct analysis of monoclonal antibody-level cross-reactivity with allelic variants feasible. ELISpot is an assay that enables that, and a recently developed multiplexed variant of ELISpot (FluoroSpot) facilitates simultaneous assessment of B-cell/antibody reactivity to several different antigens. However, serological analysis often does not allow the distinction between true cross-reactivity (one antibody recognizing multiple antigen variants) and apparent cross-reactivity (presence of multiple variant-specific antibodies), as it requires analysis at the single B-cell/monoclonal antibody level. The issue of antibody cross-reactivity is of central importance in immunology, and not least in protective immunity to Plasmodium falciparum malaria, where key antigens show substantial allelic variation (polymorphism).
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